Sodium Channel Activity Research Articles

The role of NaV channels in synaptic transmission after axotomy in a microfluidic culture platform

Voltage gated sodium channels are key players in aberrant pain signaling and sensitization of nociceptors after peripheral nerve injury. The extent to which sodium channel activity after injury contributes to synaptic transmission at the first pain synapse however remains unclear. To investigate the effect of axotomy on synaptic transmission between dorsal root ganglia neurons and dorsal horn neurons, we reconstructed the first pain synapse in a novel microfluidic based compartmentalized cell culture system, which recapitulates the connectivity of peripheral pain signaling. We show that following axotomy of the distal axons, inhibition of NaV1.7 and NaV1.8 sodium channels in incoming presynaptic DRG axons is no longer sufficient to block activation of these axons and the resulting synaptic transmission to dorsal horn neurons. We found that blockade of NaV1.6 activity is highly effective in reducing activation of incoming axons contributing to synaptic transmission after axotomy of DRG neurons. The microfluidic culture system described here offers an in vitro platform to recapitulate and study the first pain synapse.

Abstract 009: Endothelial Epithelial Sodium Channel Promotes Intrinsic Cardiomyocyte Stiffness and Diastolic Dysfunction in Response to a Diet Induced Obesity in Female Mice

Obesity is associated with a metabolic cardiomyopathy characterized by diastolic dysfunction with preserved systolic function. Although premenopausal non-obese women show relative protection against development of metabolic cardiomyopathy and cardiovascular disease compared to men, this relationship is lost in obese women. Diastolic dysfunction in such cardiomyopathy is caused not only by extrinsic extracellular remodeling and interstitial fibrosis but also through alterations in intrinsic stiffness of cardiomyocytes. One mechanism by which intrinsic cardiomyocyte stiffness is increased in obesity is decreased coronary endothelial cell production of nitric oxide. We have described a clinically relevant diet induced obesity murine model that exhibits increased vascular stiffness associated with cardiac dysfunction. In this model, female mice have high plasma aldosterone levels and increased mineralocorticoid receptor (MR) expression in both the vasculature and heart. One of the mechanisms by which MR activation promotes endothelial stiffness is through increased expression and activation of epithelial sodium channel (ENaC) in ECs (EnNaC). In this study, we tested the hypothesis that specific deletion of EnNaC ameliorates increases in endothelial stiffness and decreases diastolic dysfunction occurring during diet induced obesity (DIO) in female mice. To produce cell specific deletion of the EnNaC gene,”floxed” EnNaC mice were serially crossed with Tie 2-Cre transgene mice. This resulted in marked suppression of EnNaC expression in ECs. Female KO mice and littermate controls were fed a diet high in fat (46%) and fructose (17.5%) for 12 to 16 weeks. Compared to mice fed a control diet (CD), whole cell sodium currents in pulmonary ECs and aortic EC stiffness were significantly increased in DIO mice and this was prevented in EnNaC KO mice with DIO. Moreover, deletion of EnNaC also prevented DIO increases in stiffness of isolated cardiomyocytes (WD, 7.43 ± 1.21 kPA, WD-KO, 3.47 ± 0.14 kPA) as well as normalizing impaired diastolic relaxation. Collectively, these findings support the notion that DIO promotes coronary ECMR mediated activation of EnNaC and associated increases in intrinsic cardiomyocyte stiffness and diastolic dysfunction.

Nuclear Factor-kappaB Gates Nav1.7 Channels in DRG Neurons via Protein-Protein Interaction.

SummaryIt is well known that nuclear factor-kappaB (NF-κB) regulates neuronal structures and functions by nuclear transcription. Here, we showed that phospho-p65 (p-p65), an active form of NF-κB subunit, reversibly interacted with Nav1.7 channels in the membrane of dorsal root ganglion (DRG) neurons of rats. The interaction increased Nav1.7 currents by slowing inactivation of Nav1.7 channels and facilitating their recovery from inactivation, which may increase the resting state of the channels ready for activation. In cultured DRG neurons TNF-α upregulated the membrane p-p65 and enhanced Nav1.7 currents within 5 min but did not affect nuclear NF-κB within 40 min. This non-transcriptional effect on Nav1.7 may underlie a rapid regulation of the sensibility of the somatosensory system. Both NF-κB and Nav1.7 channels are critically implicated in many physiological functions and diseases. Our finding may shed new light on the investigation into the underlying mechanisms.

Vaporized E-Cigarette Liquids Induce Ion Transport Dysfunction in Airway Epithelia.

Cigarette smoking is associated with chronic obstructive pulmonary disease and chronic bronchitis. Acquired ion transport abnormalities, including cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction, caused by cigarette smoking have been proposed as potential mechanisms for mucus obstruction in chronic bronchitis. Although e-cigarette use is popular and perceived to be safe, whether it harms the airways via mechanisms altering ion transport remains unclear. In the present study, we sought to determine if e-cigarette vapor, like cigarette smoke, has the potential to induce acquired CFTR dysfunction, and to what degree. Electrophysiological methods demonstrated reduced chloride transport caused by vaporized e-cigarette liquid or vegetable glycerin at various exposures (30 min, 57.2% and 14.4% respectively, vs. control; P < 0.0001), but not by unvaporized liquid (60 min, 17.6% vs. untreated), indicating that thermal degradation of these products is required to induce the observed defects. We also observed reduced ATP-dependent responses (-10.8 ± 3.0 vs. -18.8 ± 5.1 μA/cm2 control) and epithelial sodium channel activity (95.8% reduction) in primary human bronchial epithelial cells after 5 minutes, suggesting that exposures dramatically inhibit epithelial ion transport beyond CFTR, even without diminished transepithelial resistance or cytotoxicity. Vaporizing e-cigarette liquid produced reactive aldehydes, including acrolein (shown to induce acquired CFTR dysfunction), as quantified by mass spectrometry, demonstrating that respiratory toxicants in cigarette smoke can also be found in e-cigarette vapor (30 min air, 224.5 ± 15.99; unvaporized liquid, 284.8 ± 35.03; vapor, 54,468 ± 3,908 ng/ml; P < 0.0001). E-cigarettes can induce ion channel dysfunction in airway epithelial cells, partly through acrolein production. These findings indicate a heretofore unknown toxicity of e-cigarette use known to be associated with chronic bronchitis onset and progression, as well as with chronic obstructive pulmonary disease severity.

Cathepsin B increases ENaC activity leading to hypertension early in nephrotic syndrome.

The NPHS2 gene, encoding the slit diaphragm protein podocin, accounts for genetic and sporadic forms of nephrotic syndrome (NS). Patients with NS often present symptoms of volume retention, such as oedema formation or hypertension. The primary dysregulation in sodium handling involves an inappropriate activation of the epithelial sodium channel, ENaC. Plasma proteases in a proteinuria‐dependent fashion have been made responsible; however, referring to the timeline of symptoms occurring and underlying mechanisms, contradictory results have been published. Characterizing the mouse model of podocyte inactivation of NPHS2 (Nphs2∆pod) with respect to volume handling and proteinuria revealed that sodium retention, hypertension and gross proteinuria appeared sequentially in a chronological order. Detailed analysis of Nphs2∆pod during early sodium retention, revealed increased expression of full‐length ENaC subunits and αENaC cleavage product with concomitant increase in ENaC activity as tested by amiloride application, and augmented collecting duct Na+/K+‐ATPase expression. Urinary proteolytic activity was increased and several proteases were identified by mass spectrometry including cathepsin B, which was found to process αENaC. Renal expression levels of precursor and active cathepsin B were increased and could be localized to glomeruli and intercalated cells. Inhibition of cathepsin B prevented hypertension. With the appearance of gross proteinuria, plasmin occurs in the urine and additional cleavage of γENaC is encountered. In conclusion, characterizing the volume handling of Nphs2∆pod revealed early sodium retention occurring independent to aberrantly filtered plasma proteases. As an underlying mechanism cathepsin B induced αENaC processing leading to augmented channel activity and hypertension was identified.

Hyperexcitability precedes motoneuron loss in the Smn2B/- mouse model of spinal muscular atrophy.

Spinal motoneuron dysfunction and loss are pathological hallmarks of the neuromuscular disease spinal muscular atrophy (SMA). Changes in motoneuron physiological function precede cell death, but how these alterations vary with disease severity and motoneuron maturational state is unknown. To address this question, we assessed the electrophysiology and morphology of spinal motoneurons of presymptomatic Smn2B/- mice older than 1 wk of age and tracked the timing of motor unit loss in this model using motor unit number estimation (MUNE). In contrast to other commonly used SMA mouse models, Smn2B/- mice exhibit more typical postnatal development until postnatal day (P)11 or 12 and have longer survival (~3 wk of age). We demonstrate that Smn2B/- motoneuron hyperexcitability, marked by hyperpolarization of the threshold voltage for action potential firing, was present at P9-10 and preceded the loss of motor units. Using MUNE studies, we determined that motor unit loss in this mouse model occurred 2 wk after birth. Smn2B/- motoneurons were also larger in size, which may reflect compensatory changes taking place during postnatal development. This work suggests that motoneuron hyperexcitability, marked by a reduced threshold for action potential firing, is a pathological change preceding motoneuron loss that is common to multiple models of severe SMA with different motoneuron maturational states. Our results indicate voltage-gated sodium channel activity may be altered in the disease process.NEW & NOTEWORTHY Changes in spinal motoneuron physiologic function precede cell death in spinal muscular atrophy (SMA), but how they vary with maturational state and disease severity remains unknown. This study characterized motoneuron and neuromuscular electrophysiology from the Smn2B/- model of SMA. Motoneurons were hyperexcitable at postnatal day (P)9-10, and specific electrophysiological changes in Smn2B/- motoneurons preceded functional motor unit loss at P14, as determined by motor unit number estimation studies.

Mucoadhesive Electrospun Patch Delivery of Lidocaine to the Oral Mucosa and Investigation of Spatial Distribution in a Tissue Using MALDI-Mass Spectrometry Imaging.

Many oral mucosal conditions cause considerable and prolonged pain that to date has been difficult to alleviate via topical delivery, and the use of injection causes many patients dental anxiety and needle-prick pain. Therefore, developing a noninjectable drug delivery system as an alternative administration procedure may vastly improve the health and wellbeing of these patients. Recent advances in the development of mucoadhesive electrospun patches for the direct delivery of therapeutics to the oral mucosa offer a potential solution, but as yet, the release of local anesthetics from this system and their uptake by oral tissue have not been demonstrated. Here, we demonstrate the fabrication of lidocaine-loaded electrospun fiber patches, drug release, and subsequent uptake and permeation through the porcine buccal mucosa. Lidocaine HCl and lidocaine base were incorporated into the electrospun patches to evaluate the difference in drug permeation for the two drug compositions. Lidocaine released from the lidocaine HCl-containing electrospun patches was significantly quicker than from the lidocaine base patches, with double the amount of drug released from the lidocaine HCl patches in the first 15 min (0.16 ± 0.04 mg) compared to that from the lidocaine base patches (0.07 ± 0.01 mg). The permeation of lidocaine from the lidocaine HCl electrospun patches through ex vivo porcine buccal mucosa was also detected in 15 min, whereas permeation of lidocaine from the lidocaine base patch was not detected. Matrix-assisted laser desorption ionization-mass spectrometry imaging was used to investigate localization of lidocaine within the oral tissue. Lidocaine in the solution as well as from the mucoadhesive patch penetrated into the buccal mucosal tissue in a time-dependent manner and was detectable in the lamina propria after only 15 min. Moreover, the lidocaine released from lidocaine HCl electrospun patches retained biological activity, inhibiting veratridine-mediated opening of voltage-gated sodium channels in SH-SY5Y neuroblastoma cells. These data suggest that a mucoadhesive electrospun patch may be used as a vehicle for rapid uptake and sustained anesthetic drug delivery to treat or prevent oral pain.

Purification and biochemical characterisation of a putative sodium channel agonist secreted from the South African Knobbly sea anemone Bunodosoma capense

Voltage gated ion channels have become a subject of investigation as possible pharmaceutical targets. Research has linked the activity of ion channels directly to anti-inflammatory pathways, energy homeostasis, cancer proliferation and painful diabetic neuropathy. Sea anemones secrete a diverse array of bioactive compounds including potassium and sodium channel toxins. A putative novel sodium channel agonist (molecular mass of 4619.7 Da) with a predicted sequence: CLCNSDGPSV RGNTLSGILW LAGCPSGWHN CKKHKPTIGW CCK was isolated from Bunodosoma capense using a modified stimulation technique to induce the secretion of the neurotoxin rich mucus confirmed by an Artemia nauplii bio-assay. The peptide purification combined size-exclusion and reverse-phase high performance liquid chromatography. A thallium-based ion flux assay confirmed the presence of a sodium channel agonist/inhibitor and purity was determined using a modified tricine SDS-PAGE system. The peptide isolated indicated the presence of multiple disulfide bonds in a tight β-defensin cystine conformation. An IC50 value of 26 nM was determined for total channel inhibition on MCF-7 cells. The unique putative sodium channel agonist initiating with a cystine bond indicates a divergent evolution to those previously isolated from Bunodosoma species.

Epithelial sodium channels in endothelial cells mediate diet-induced endothelium stiffness and impaired vascular relaxation in obese female mice

ObjectiveMineralocorticoid receptor activation of the epithelial sodium channel in endothelial cells (ECs) (EnNaC) is accompanied by aldosterone induced endothelial stiffening and impaired nitric oxide (NO)-mediated arterial relaxation. Recent data support enhanced activity of the alpha subunit of EnNaC (αEnNaC) mediates this aldosterone induced endothelial stiffening and associated endothelial NO synthase (eNOS) activation. There is mounting evidence that diet induced obesity diminishes expression and activation of AMP-activated protein kinase α (AMPKα), sirtuin 1 (Sirt1), which would be expected to lead to impaired downstream eNOS activation. Thereby, we posited that enhanced EnNaC activation contributes to diet induced obesity related increases in stiffness of the endothelium and diminished NO mediated vascular relaxation by increasing oxidative stress and related inhibition of AMPKα, Sirt1, and associated eNOS inactivation. Materials/MethodsSixteen to twenty week-old αEnNaC knockout (αEnNaC−/−) and wild type littermate (EnNaC+/+) female mice were fed a mouse chow or an obesogenic western diet (WD) containing excess fat (46%) and fructose (17.5%) for 16 weeks. Sodium currents of ECs, endothelial stiffness and NO mediated aortic relaxation were examined along with indices of aortic oxidative stress, vascular remodeling and fibrosis. ResultsEnhanced EnNaC activation-mediated WD-induced increases in sodium currents in isolated lung ECs, increased endothelial stiffness and impaired aortic endothelium-dependent relaxation to acetylcholine (10−9–10−4 mol/L). These abnormalities occurred in conjunction with WD-mediated aortic tissue oxidative stress, inflammation, and decreased activation of AMPKα, Sirt1, and downstream eNOS were substantially mitigated in αEnNaC−/− mice. Importantly, αEnNaC−/− prevented WD induced increases in endothelial stiffness and related impairment of endothelium-dependent relaxation as well as aortic fibrosis and remodeling. However, EnNaC signaling was not involved in diet-induced abnormal expression of adipokines and CYP11b2 in abdominal aortic perivascular adipose tissue. ConclusionThese data suggest that endothelial specific EnNaC activation mediates WD-induced endothelial stiffness, impaired eNOS activation, aortic fibrosis and remodeling through increased aortic oxidative stress and increased inflammation related to a reduction of AMPKα and Sirt 1 mediated eNOS phosphorylation/activation and NO production.

MrgprX1 Mediates Neuronal Excitability and Itch Through Tetrodotoxin-Resistant Sodium Channels.

In this study, we sought to elucidate the molecular mechanism underlying human Mas-related G protein-coupled receptor X1 (MrgprX1) mediated itch sensation. We found that activation of MrgprX1 by BAM8-22 triggered robust action potential discharges in dorsal root ganglion (DRG) neurons. This neuronal excitability is not mediated by Transient receptor potential (TRP) cation channels, M-type potassium channels, or chloride channels. Instead, activation of MrgprX1 lowers the activation threshold of TTX-resistant sodium channels and induces inward sodium currents. These MrgprX1-elicited action potential discharges can be blocked by Pertussis toxin (PTX) and a Gβγ inhibitor - Gallein. Behavioral results showed that Nav1.9 knockout but not Trpa1 knockout significantly reduced BAM8-22 evoked scratching behavior. Collectively, these data suggest that activation of MrgprX1 triggers itch sensation by increasing the activity of TTX-resistant voltage-gated sodium channels.

Association of Urinary Plasminogen-Plasmin with Edema and Epithelial Sodium Channel Activation in Patients with Nephrotic Syndrome

Background: Previous animal experiments and small human studies suggest that urinary plasmin can activate the epithelial sodium channel (ENaC) and contribute to sodium retention in nephrotic syndrome (NS), but this however is not well studied in clinical settings, and its relevance to edema formation is not well characterized in humans. We have investigated the association between urinary plasmin and clinical phenotypes in a large group of patients with NS from multiple etiologies, aiming to assess the role of urinary plasmin in sodium handling and edema formation. Methods: Two hundred and three NS patients with urine and blood samples were divided into mild and severe symptom groups based on their edema severity. Twenty six of them had serial samples collected during the course of immunosuppressive therapy. The plasminogen-plasmin level and other key parameters were assayed, and their association with clinical manifestations were analyzed. Results: One hundred and one of the 203 patients had renal biopsies performed, the results of which had included all the common types of primary NS and various types of secondary NS. Quantitative comparison and multivariate logistic regression analysis identified urinary plasminogen-plasmin to creatinine ratio (uPLG-PL/C), serum albumin, D-Dimer, and cardiac dysfunction history, but not albuminuria or 24-h urine protein, as independent risk factors for edema (p < 0.01). In patients who were treated and had serial samples, a decrease in uPLG-PL/C was identified as an independent influencing factor of edema remission (p < 0.01). Finally, the urinary fractional excretion of sodium (FENa) in patients was inversely correlated with the fractional excretion of potassium (FEK; p< 0.001), and FEK/FENa ratio was positively correlated with uPLG-PL/C (p < 0.001), suggesting a close association between uPLG-PL and ENaC activation. Conclusions: Our study identifies uPLG-PL abundance as an independent influencing factor of edema in adult NS patients, and supports the conclusion that plasmin-dependent ENaC activation is an important pathophysiological mechanism of sodium retention and edema formation in humans with NS.

Abstract 2001: Inhibiting voltage-gated sodium channel activity in medullary thyroid cancer using small molecule compounds

Abstract Medullary thyroid cancer (MTC) is a type of neuroendocrine cancer (NE) with increasing incidence worldwide. Compared with well-differentiated thyroid cancers, distant metastases are more commonly observed in MTC patients, ranging from 7-23%. The inability to treat metastatic MTC contributes to a decreased patient survival rate. Currently, at least nine mammalian voltage-gated sodium channels (VGSCs) named Nav1.1-Nav1.9 have been identified as potential safe drug targets for treating metastasis in various cancers. Herein, we investigate the effects of targeting VGSCs in MTC using small molecule inhibitors. Two MTC cell lines, TT and MZ, were treated with four compounds and the changes in mRNA and protein expression of VGSC isoform Nav1.7 were assessed through RT-PCR and western blotting. Treatment by one of the compounds exhibited a 28-fold decrease in mRNA expression of VGSC isoform 1.7. Furthermore, a cell proliferation assay, proliferation assay (MTT) a MTT, assay to study cell proliferation after treatment showed decreasing cell viability resulted from the treatment. and Additionally, morphological changes characteristic of apoptosis in fluorescently labeled MTC cell lines after 48 hours of treatment with VGSC inhibitory compounds were observed. A Boyden chamber assay was also performed on MTC cell lines to examine changes in migration and invasion after administering the potential inhibitory compounds. This study has generated preliminary evidence that voltage-gated sodium channels (VGSCs) may indeed be potential targets for treating metastatic MTC. The results may be translated to future studies focused on controlling metastasis through specified drug treatment in MTC, leading to the limitation of cancer progression by means of targeting VGSCs. Citation Format: Paras Ahuja, Rachael Guenter, Jaden Cowan, Yazen Shihab, Jason Whitt, Herbert Chen, Sadanandan Velu, Renata Jaskula-Sztul. Inhibiting voltage-gated sodium channel activity in medullary thyroid cancer using small molecule compounds [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2001.

Analgesic effects of amiodarone in mouse models of pain.

Purpose: Although amiodarone is classified as a Vaughan-Williams class Ⅲ antiarrhythmic drug, it has inhibitory effects on voltage-gated sodium and calcium channels and on β-adrenergic receptors. Given these pharmacological profiles, amiodarone may have analgesic properties. Most patients who are prescribed amiodarone possess multiple cardiovascular risk factors. Despite the fact that pain plays a crucial role as a clinical indicator of cardiovascular events, the effects of amiodarone on pain have not been investigated. The aim of the current study was to investigate the analgesic effects of amiodarone by using mouse models of pain in an effort to elucidate underlying mechanisms.Methods: Adult male C57B6 mice received single bolus intraperitoneal injections of amiodarone at doses of 25, 50, 100, and 200 mg/kg, while the mice in the control group received only normal saline. The analgesic effects of amiodarone were evaluated using the acetic acid-induced writhing test, formalin test, and tail withdrawal test. In addition, the potassium channel opener NS1643, voltage-gated sodium channel opener veratrine, calcium channel opener BAYK8644, and selective β-adrenergic agonist isoproterenol were used to uncover the underlying mechanism.Results: During the acetic acid-induced writhing test, formalin test, and tail withdrawal test, amiodarone induced analgesic responses in a dose-dependent manner. The analgesic effects of amiodarone were abolished by veratrine but not by NS1643, BAYK8644, or isoproterenol.Conclusion: Amiodarone induced analgesic responses in a dose-dependent manner, likely by blocking voltage-gated sodium channels. These results indicate that clinical doses of amiodarone can affect nociception and may mask or attenuate pain induced by acute cardiovascular events.

Urokinase-type plasminogen activator (uPA) is not essential for epithelial sodium channel (ENaC)-mediated sodium retention in experimental nephrotic syndrome.

In nephrotic syndrome, aberrantly filtered plasminogen (plg) is converted to active plasmin by tubular urokinase-type plasminogen activator (uPA) and thought to lead to sodium retention by proteolytic activation of the epithelial sodium channel (ENaC). This concept predicts that uPA is an important factor for sodium retention and that inhibition of uPA might be protective in nephrotic syndrome. Activation of amiloride-sensitive currents by uPA and plg were studied in Xenopus laevis oocytes expressing murine ENaC. In doxorubicin-induced nephrotic mice, uPA was inhibited pharmacologically by amiloride and genetically by the use of uPA-deficient mice (uPA-/- ). Experiments in Xenopus laevis oocytes expressing murine ENaC confirmed proteolytic ENaC activation by a combination of plg and uPA which stimulated amiloride-sensitive currents with concomitant cleavage of the ENaC γ-subunit at the cell surface. Treatment of nephrotic wild-type mice with amiloride inhibited urinary uPA activity, prevented urinary plasmin formation and sodium retention. In nephrotic mice lacking uPA (uPA-/- ), urinary plasmin formation from plg was suppressed and urinary uPA activity absent. However, in nephrotic uPA-/- mice, sodium retention was not reduced compared to nephrotic uPA+/+ mice. Amiloride prevented sodium retention in nephrotic uPA-/- mice which confirmed the critical role of ENaC in sodium retention. uPA is responsible for the conversion of aberrantly filtered plasminogen to plasmin in the tubular lumen in vivo. However, uPA-dependent plasmin generation is not essential for ENaC-mediated sodium retention in experimental nephrotic syndrome.

SAT-LB011 Role of Endothelium Epithelial Sodium Channel in Arterial Stiffness

Excessive epithelial sodium channel activation in endothelium (EnNaC) increase oxidative stress and inflammation with associated cardiovascular abnormalities. Our recent data has shown that activation of EnNaC mediates aortic endoplasmic reticulum stress, redox oxidative stress, expression of pro-inflammatory cytokines, and aortic remodeling. These abnormalities are potentially related to abnormal expression and activation of sirtuin 1 (Sirt1) and AMP-activated protein kinase (AMPK) in metabolic disorders such as obesity. We hypothesized that EnNaC mediates Western diet (WD)-induced endothelium and aortic stiffness by increasing aortic oxidative stress and associated oxidative stress related inhibition of Sirt1 and AMPK expression and activation. Accordingly, sixteen to twenty week-old EnNaC-/- and wild type littermate female mice were fed a mouse chow or an obesogenic WD containing excess fat (46%) and fructose (17.5%) for 16 weeks. EnNaC activation mediated WD-induced increase of Na+ currents in isolated lung endothelial cells, reduction of phosphorylation of endothelial nitric oxide synthase (eNOS), as well as impairment of aortic endothelium-dependent relaxation to acetylcholine (10-9-10-4 mol/L) in a wire myograph. The expression and activation of Sirt1 and AMPK were inhibited in WD EnNaC+/+ mice and these abnormalities were prevented in EnNaC-/- mice. Importantly, EnNaC specific KO prevented WD induced aortic oxidative stress and related endothelium stiffness and impairment of endothelium (NO)-dependent relaxation. These data suggest that endothelial specific EnNaC activation mediates WD-induced aortic oxidative stress, decreased expression and activation of Sirt1, AMPK, eNOS, as well as endothelial and aortic vessel stiffness. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Effects of skin cooling on intra-articular knee temperature in healthy subjects

Purpose: Cooling is widely used to control pain associated with acute injury and surgery involving the knee. Cooling may affect pain by exerting effects on the inflammatory cascade, transduction channels in nociceptors, sodium channel activity in neurons, or may suppress pain through masking effects mediated by central mechanisms. CNTX-4975, a highly purified, synthetic trans-capsaicin, is under investigation in phase 3 trials to determine efficacy and safety when administered via intra-articular (IA) injection in patients with moderate to severe knee osteoarthritis pain. Cooling has been demonstrated to decrease pain associated with topical capsaicin without interfering with the therapeutic response. In other studies, it has been determined that cooling markedly decreases the initial pain associated with capsaicin injection. Here we report temperature variations of both the skin and the IA space in healthy volunteers using 2 different cooling techniques currently used in clinical practice Methods: Two knee cooling techniques were used in healthy volunteers: an ice pack placed on the patella of one knee, and a commercially available circumferential circulating ice water wrap (CCIWW) placed on the skin surrounding the other knee. To reduce bias from order effects, 3 subjects received the CCIWW on the left knee and ice pack on the right, and the other 2 subjects received ice pack cooling on the left knee and CCIWW on the right. Two temperature probes were used for each knee: an IA probe and a skin probe placed adjacent to the superior pole of the patella under the cooling apparatus. After baseline temperature measurements in the left knee, cooling was applied for 15 minutes, at which point subjects received an IA injection of 15 mL of 2% lidocaine without epinephrine (to simulate the procedures used with the CNTX-4975 IA injection procedure). IA CNTX-4975 was not administered. Cooling was restarted and continued up to a total of 120 minutes. The process was repeated for the right knee after a rest period (≤4 hours) following left knee measurements. Temperatures within the knee were measured at approximately 5-minute intervals from probe placement until removal. Probe removal was approximately 30 minutes following cessation of cooling. Subjects were asked to rate their pain using a numeric pain rating scale (0-10; 0=no pain; 10=worst imaginable pain) at various time points throughout the procedure. At subsequent visits, knees were inspected for any injection site issues, and adverse events were recorded. Results: Five healthy male subjects were enrolled; mean age, 30 years. All subjects completed the study for both knees. The Figure shows the mean temperature change at the skin surface and in the joint over time under the 2 cooling conditions. The mean baseline skin and IA temperatures were approximately 29°C and 33°C, respectively. The decrease in skin temperature was similar between techniques for the first 40 minutes, reaching a temperature of approximately 22°C. With the ice pack, the skin temperature plateaued after 50 to 60 minutes and had a temperature of 19°C at 140 minutes when cooling was removed. In contrast, with the CCIWW technique, the skin temperature continued to decrease to 14°C at 140 minutes. The ice pack did not have an appreciable effect on the IA temperature at any point. With the CCIWW technique, 40 minutes after the start of cooling, the mean IA knee temperature had decreased by approximately 2°C, and by 140 minutes had decreased by approximately 10°C to a temperature of 23.9°C (95% CI, 22.6-25.2). Even after the cooling was stopped, there was a delay before the IA temperature began to rise. Subjects reported no pain from either the skin or the joint. The procedures overall were well tolerated and there were no complications. Conclusions: The baseline knee IA temperature under ambient conditions was considerably below core temperature and approximately 33°C. The mean baseline skin temperature overlying the joint was approximately 29°C. Circumferential cooling provided by the CCIWW technique was superior to cooling with the ice packs, resulting in a 9°C decrease in skin temperature and an approximate 10°C decrease in joint temperature over 140 minutes. Although there was initially a lag, decreases in joint and skin temperature were temporally parallel. The thermal inertia was such that the IA temperature decreased even after cessation of cooling before it began to rise. Effective joint cooling appears to favor a method that provides circumferential cooling of the joint. Subsequent studies will determine how procedural pain from trans-capsaicin injection into an osteoarthritic knee relates to joint temperature.

Local Anesthetics Induce Cardiac Toxicity Through Distinct Effects on Calcium Dynamics: Implications For Clinical Prevention

INTRODUCTIONThe clinically safe dose of local anesthetics is limited by their induction of potentially lethal cardiac arrhythmias, which occurs in ~50,000 people in the US per year. While this drug toxicity is often attributed to non‐specific block of cardiac sodium channels, studies on isolated channels fail to show any clinically relevant difference between more harmful (i.e. bupivacaine, BUP) and less harmful (i.e. ropivacaine, ROP) anesthetics. We sought to determine the mechanisms of toxicity and arrhythmogenesis for BUP and ROP, revealing prevention and treatment strategies to improve clinical safety.METHODSWe cultured beating 2D‐tissues of human iPSC‐derived cardiomyocytes and treated them with clinically relevant concentrations of BUP or ROP. We then analyzed changes to ion and contractile dynamics using the xCELLigence RTCA Cardio and the cytosolic calcium dye FLIPR5. As an inotrope, calcium was tested for its ability to recover contractile dysfunction. Our in vitro findings were validated in a rat model of BUP toxicity. Anesthetized animals were infused with either saline or CaCl2 (10 mg/kg over 5 min) in the tail vein, followed by either 2 mg/kg/min BUP or ROP, until asystole. Cardiac activity was monitored via ECG and invasive arterial blood pressure recordings, and analysed using non‐linear regression incorporating the effects of drug treatment, sampling time, and animal variability.RESULTSIn human iPSC cardiomyocytes, both anesthetics affected contractility, beat rate, rhythm, excitation‐contraction coupling, and sodium channel activity as expected, with BUP causing a greater adverse effect. BUP, but not ROP, additionally delayed calcium wave duration by 20% and reduced overall calcium flux by 66%. Interestingly, CaCl2 co‐treatment with BUP abolished aberrancy in all toxicity metrics, where CaCl2 co‐treatment with ROP exacerbated toxicity. Further investigation into intracellular calcium dynamics revealed BUP‐specific defects in diastolic and systolic calcium flux, which were mitigated by CaCl2 co‐treatment. Our in vivo model validated these findings, with ROP showing less toxicity than BUP in all observed metrics. CaCl2 pre‐treatment mitigated the BUP‐mediated decline in beat rate and diastolic arterial pressure, but had no effect, or exacerbated outcomes, in ROP‐treated rats. Furthermore, CaCl2 pre‐treatment reduced arrhythmia onset and QRS widening cased by BUP, but exacerbated these arrhythmia metrics and reduced survival with ROP.CONCLUSIONSWe found that BUP, but not ROP, caused a baseline dysfunction in calcium dynamics in human iPSC‐derived cardiomyocytes. Calcium pre‐treatment prevented BUP, but exacerbated ROP cardiotoxicity in both in vitro and in vivo models. Together, our results implicate distinct calcium signaling and dynamics in eliciting the higher clinical cardiotoxicity of the BUP local anesthetic, and show how calcium can be used to protect against the arrhythmogenic effects of BUP overdose.Support or Funding InformationCanadian Institutes of Health ResearchThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inducible renal‐tubule insulin receptor (IR) knockout affects sodium transporter/channel expression and activity in a sex‐dependent manner

Insulin has been demonstrated to be natriferic in thick ascending limb (TAL), distal convoluted tubule (DCT), and collecting duct (CD). Direct activation of sodium (Na+) transporters and channels by the insulin receptor (IR) likely underlies this effect. To elucidate the role of tubular IR on blood pressure (BP) and apical transporter/channel activity at these sites, we knocked out (KO) IR from the renal tubule, in male (M) and female (F) mice (C57Bl6 background) using a tetracycline‐inducible system. Doxycycline (DOX) was fed to all mice ad libitum (625 mg/kg·diet) for two weeks to induce KO in homozygously IR floxed mice harboring both inducible Cre and paired box gene 8 (PAX8) promoter transgenes. WT mice were negative for Cre, PAX8 or both transgenes. BP and sensitivity to select diuretics was assessed in the pre‐DOX and post‐DOX states. Four weeks post‐DOX, mice were euthanized 4 hours after an acute injection of insulin (0.5 U/kg·bw) and blood and kidneys collected for analysis. Two‐way ANOVA (sex X genotype) revealed DOX marginally reduced kidney weight (p = 0.059) and increased blood potassium (K+, p = 0.02). These differences were abrogated in F. Final blood glucose was also lower in F (p = 0.001) and in KO mice (p = 0.016). Pre‐Dox BP (mm Hg) measured by tail‐cuff plethysmography was similar in M (103.1 ± 3.2) and F (100.2 ± 6.0). DOX caused a fall in MAP (~ 5 mm Hg) in F, but not M KO mice. Natriuretic responses to furosemide (FUR), thiazide (TZD), and benzamil (BNZ) were used as indexes of Na‐K‐2Cl cotransporter (NKCC2), Na‐Cl cotransporter (NCC), and epithelial sodium channel (ENaC) activity, respectively, and were all marginally‐to‐significantly higher in F versus M mice in the pre‐Dox state (18, 300, and 44% higher, respectively), suggesting greater basal activity of these Na+ reabsorptive routes in F (n = 12–17/sex). After DOX, there was a significant interaction (p = 0.010, sex X genotype) for the response to FUR, with M KO responding with an increase, and F KO, with a decrease. The response to TZD tended to increase in the M KO, but decrease in the F KO (relative to same sex WT). The response to BNZ remained higher in the F, and was slightly increased in both sexes by KO (~20–50%). In cortex homogenates (CTXH), western blotting revealed band density for the β‐subunit of IR decreased on average 60% with no sex difference. F had significantly higher band density for α‐ENaC (90‐kDa band), as compared to M (p &lt; 0.0001), and KO led to a significant interactive term in that band density was decreased in F, but increased in M. Both sexes showed reduced band densities for β‐ and γ‐ENaC (85 kDa) and outer medullary NKCC2 in KO. NCC band density was not affected by KO, but significantly higher in F (p &lt; 0.0001). Taken together these results demonstrate differential sensitivity between the sexes to renal tubular IR KO in regard to homeostatic control of Na+ and K+ handling and BP. KO led to modest reductions in kidney weight and transporter/channel expression levels, supporting the role for insulin in growth and maintenance of cell size and differentiation. Little to no reduction in measurable activity could result from compensation, which appeared more effective in males.Support or Funding InformationAmerican Diabetes Association (MF)Georgetown University Medical CenterThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The iPSC‐derived human dopamine neurons from a healthy subject and a patient with α‐synuclein triplication reveal novel therapeutic target

Parkinson's disease (PD) is characterized by progressive loss of dopamine (DA) neurons in the substantia nigra (SN-A9). α-synuclein has been genetically implicated in familial and sporadic PD and is associated with PD susceptibility, progression and pathology. A cardinal feature of neurons that separates them from nearly all other cell types is their excitability. Multiple channels and transporters regulate the excitability of DA neurons. DA neurons are uniquely defined by expression of Dopamine Transporter (DAT), D2 autoreceptor and GIRK, where they govern the excitability of DA neurons, and thus DA neurotransmission. To determine the impact of α-synuclein on the activity of DA neurons, we differentiated induced pluripotent stem cells (iPSC) from PD patients with α-synuclein triplication and the healthy sibling. Two sets of iPSCs used in our studies: iPSCs with triplication of α-synuclein (AST), and iPSCs with normal α-synuclein (NAS) levels. The A9 human-like DA neurons were differentiated and identified by expression of Tyrosine Hydroxylase (TH), GIRK2, VMAT2, DAT, Nurr1, FOXA2, as phenotypic markers for DA neurons. The expression of α-synuclein and Parkin, two PD-associated proteins were higher in the AST-derived human-like DA neurons as compared to the NAS-derived human-like DA neurons. We found significant morphological differences between neurons with normal α-synuclein levels (NAS) vs. neurons with α-synuclein triplication (AST). The AST neurons exhibited significantly larger soma, short neurites and lower D2R and GIRK expression (P<0.005). Importantly, our differentiation protocol resulted in spontaneously firing human-like DA neurons. Whole-cell voltage-clamp recordings revealed no difference in the overall activity of sodium and potassium channels in the AST and NAS DA neurons ((P>0.05). Consistent with our previous report in mice DA neurons (Lin et al., 2016), the majority of NAS-derived DA neurons exhibited a mixture of single spikes and small burst activity with an underlying “pacemaker-like” periodicity. Whereas, ~90% of AST-derived DA neurons exhibited a unique firing pattern of spontaneous firing activity with a pause between subsequent broadbrimmed burst clusters on a high depolarized plateau (up state) (P<0.05). We found, in the AST-derived DA neurons, activation of D2R or GIRK channels reduced the amplitude and the width of the up state and suppressed the firing frequency, suggesting α-synuclein triplication disrupts firing frequency of DA neurons and thus DA transmission via dysregulation of D2R and GIRK channels. Morphological differences and their measurements after 4 months differentiation of NAS and AST dopamine neurons Our differentiation protocol produced spontaneously active DA neurons. Increased α-Syn dysregulated the spontaneous firing activity of AST DA neurons Quinpirole activation of D2R restores endogenous AST-derived human-like DA neurons This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High mobility group box‐1 increases epithelial sodium channel activity and inflammation in small airway epithelial cells

In Cystic Fibrosis (CF) patients, inherited defects in anion transport leads to hyperactive epithelial Na+ channel (ENaC) activity. ENaC dysfunction in CF lung is known to cause airway surface liquid dehydration. Thick and dry mucus, in turn, leads to an inflammatory lung phenotype that often occurs without detection of infection in CF patients. Many investigative groups have reported that airway tissue and fluid from murine models exhibiting the CF lung phenotype (Scnn1b‐Tr mice), as well as CF patients, have increased levels of high mobility group box‐1 (HMGB‐1). HMGB‐1 is a cytokine mediator of inflammation and is a biomarker for severity of CF lung disorder. Herein, we hypothesize that HMGB‐1 plays an important role in the pathogenic regulation of ENaC, inflammation, and fibrosis in sterile airways. To begin testing this hypothesis, we measured transepithelial sodium current (Isc) and single channel ENaC open probability (Po) in human small airway epithelial cells (SAEC) in the presence or absence of 1 mg/mL human HMGB‐1 peptide. Electrophysiological measurements show that HMGB‐1 significantly increased amiloride‐sensitive ENaC Isc from −9.7±1.4 μA/cm2 to −18.4±2.9 μA/cm2 in confluent monolayers of SAEC (n=12; p=0.01) within 30 min. Subsequent amiloride treatment of the same SEAC monolayers decreased Isc 71%. Single channel measurements were conducted in primary SAEC obtained from 4 separate donor lungs in the cell‐attached configuration; HMGB‐1 increased ENaC Po from 0.18±0.02 to 0.35±0.04 (n=17; p&lt;0.01) in single channel analysis. Mean absolute total cell counts were significantly higher in bronchoalveolar lavage fluid (BALF) from mice intraperitoneally (IP) injected with HMBG‐1 (10 mg in 1 mL saline/kg) vs vehicle injected mice. Total immune cell counts were 27.4×103± 8.8×103 (in HMGB‐1) vs. 5.2×103±1.3X103 (in vehicle) injected mice (n=8 counts from 2 independent studies; p&lt;0.05). Masson's Trichrome labeling of IP injected mice show that HMGB‐1 significantly increases pulmonary fibrosis (see Fig 1). Flow cytometric analysis of HMGB‐1 and vehicle injected mice showed significant increase in IL‐1β, IL‐10, IL‐6, IL‐27, IL‐17A, IFN‐β, and GM‐CSF (n=3; p&lt;0.05; see Fig 2). Together, our data suggests that HMGB‐1 plays an important role in the CF lung phenotype; additional studies are underway to strengthen these observations. Impact: Successful completion of our work can lead to novel therapies needed to counteract ENaC dysfunction and inflammation in the CF lung.Support or Funding InformationThis work was supported by NIH 1 R01 HL137033‐01A1 awarded to MH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.